Spermatogenesis is regulated by a cascade of regulatory events involving activation of a robust transcription program in spermatocytes. Understanding the mechanisms that control this program is critical for identification of generic defects that disrupt spermatogenesis and, ultimately, for correcting such defects. Previous studies suggested that these mechanisms involve both testesspecific components of the core transcription apparatus, and gene-specific transcription factors. However, only a few of such regulators essential for spermatogenesis have been described to date, and the mechanisms by which they control spermatocyte-specific transcription are not fully understood. Recently, we identified Modulo as the first Drosophila gene-specific factor required for expression of the testes-specific genes, and for male fertility. Modulo directly binds to the target promoters and is involved in regulatory and physical interactions with the testes-specific components of the core transcription apparatus (tTAFs). In addition, Modulo controls expression of a group of transcription factors in testes. These findings support our hypothesis that Modulo acts as a key regulator of the transcriptional network in male germline. To test this hypothesis and characterize the mechanism(s) by which Modulo and the downstream Modulo-dependent transcription factors (MDTFs) regulate gene expression in male germline, we propose two Specific Aims. Under the first Aim, the regulatory interactions between Modulo and tTAFs in activation of spermatocyte-specific genes will be determined using combination of transgenic approach and biochemical assays. Under the second Aim, we will determine the role of MDTFs in spermatogenesis and in testis-specific transcription by ablating these factors in male germline using RNAi-transgenic and genetic approaches. The power of Drosophila experimental system and the rich background of previous research on Drosophila spermatogenesis will greatly facilitate the proposed analysis. Taking into consideration that the molecular mechanisms of testes-specific gene regulation show striking similarity between Drosophila and mammals and all of the transcriptional regulators analyzed here are evolutionarily conserved, we expect our Drosophila studies to provide direct mechanistic insights into mammalian spermatogenesis. This research will advance understanding the infertility and its underlying causes, and will provide a strong foundation for developing the tools to manipulate male germline development. In addition, these studies will have a broad impact on the studies of gene regulation in development